Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase
The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of (/sup 14/C)glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.
- Research Organization:
- Univ. of California, San Diego, La Jolla (USA)
- OSTI ID:
- 6500250
- Journal Information:
- Biochemistry; (United States), Vol. 27:19
- Country of Publication:
- United States
- Language:
- English
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ASPARTIC ACID
BIOLOGICAL FUNCTIONS
ENZYME INHIBITORS
BIOCHEMICAL REACTION KINETICS
AMP
CARBON 14 COMPOUNDS
CATALYSIS
CHYMOTRYPSIN
CROSS-LINKING
GLYCINE
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR STRUCTURE
PEPTIDES
PHOSPHOTRANSFERASES
SWINE
TRACER TECHNIQUES
TRYPSIN
AMINO ACIDS
ANIMALS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DOMESTIC ANIMALS
ENZYMES
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HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERIZATION
PROTEINS
REACTION KINETICS
SEPARATION PROCESSES
SERINE PROTEINASES
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550201* - Biochemistry- Tracer Techniques